UV-Visible Spectrophotometer (UV-Vis)
UV-Visible spectroscopy measures absorbance of ultraviolet and visible light by a sample. It is widely used in pharmaceutical, chemical and environmental laboratories.
Working Principle
Based on Beer-Lambert Law: Absorbance is proportional to concentration and path length.
Main Components
- Light Source (Deuterium / Tungsten)
- Monochromator
- Sample Cell (Cuvette)
- Detector (Photodiode / PMT)
ISO 17025 Relevance
Requires wavelength calibration, absorbance verification, and uncertainty estimation.
Part of Laboratory Engineering Hub
UV-Visible spectroscopy is one of the core analytical techniques within our complete laboratory analyzer engineering framework.
Explore the full Laboratory Engineering reference covering GC, HPLC, UV-Vis, ICP, Karl Fischer and Titration: Laboratory Analyzers – Engineering Fundamentals
Beer-Lambert Law – Detailed Explanation
A = ε × b × c
Where:
A = Absorbance
ε = Molar absorptivity (L·mol⁻¹·cm⁻¹)
b = Path length (cm)
c = Concentration (mol/L)
Absorbance is directly proportional to concentration and path length under ideal linear conditions.
Worked Calculation Example
Measured absorbance = 0.850 Path length = 1 cm Molar absorptivity (ε) = 17000 L·mol⁻¹·cm⁻¹
c = A / (ε × b)
c = 0.850 / (17000 × 1) = 5.0 × 10⁻⁵ mol/L
This value must fall within validated linear range for ISO 17025 compliance.
Calibration Curve & Linearity
In laboratory validation, multiple standards are prepared and plotted as:
Absorbance vs Concentration
Acceptance criteria typically require:
- Correlation coefficient (R² ≥ 0.995)
- Repeatability within acceptable %RSD
- Blank correction validation
Troubleshooting Logic Flow
- No signal → Lamp failure
- Noise → Detector instability
- Negative absorbance → Blank mismatch
- Low sensitivity → Wavelength misalignment
ISO 17025 Technical Control Points
- Wavelength accuracy verification (Holmium filter)
- Photometric accuracy check
- Stray light test
- Repeatability check (%RSD)
- Uncertainty budget documentation